The Association of Factor VIII With von Willebrand Factor

PETE LOLLAR, M.D., Division ofHematology and Oncology, Emory University School ofMedicine, Atlanta, Georgia Factor VIII (FVIII) and von Willebrand factor (vWF) are plasma glycoproteins that circulate as a tightly associated complex. Because they tend to copurify during procedures designed to isolate the biologic activities associated with them, their identity as distinct entities became unequivocally established only during the past 10 years. Improved procedures for the isolation ofFVIII, the deduction ofthe amino acid sequences of FVIII and vWF by using molecular cloning techniques and by direct sequencing, and the use of a variety of biophysical and immunochemical techniques have enhanced the understanding ofthe FVIII-vWF association. Each subunit ofmultimeric vWF potentially can bind a single heterodimeric FVIII molecule, although in vivo most of these binding sites are empty. The binding ofFVIII to vWF is primarily, if not exclusively, mediated by the light chain of FVIII to the amino-terminal region of the vWF subunit. Cleavage of a fragment from the amino-terminal region of the FVIII light chain by thrombin results in rapid dissociation ofthe FVIII-vWF complex, a process that apparently is necessary for development of procoagulant activity. Whether this cleavage is needed for the activation of FVIII in the absence of vWF is controversial. The extracellular association ofFVIII with vWF may be necessary for efficient secretion ofFVIII from its cell of origin. The thermodynamics, kinetics, and nature of the molecular contacts involved in the interaction have not been studied. The association of FVIII with vWF prolongs the lifetime of FVIII in plasma. Whether the FVIII-vWF interaction has other functional roles, such as restricting the location of procoagulant activity, remains unknown.

Factor VIII (FVIII) and von Willebrand factor (vWF) can be defined as the factors that correct the plasmatic defect in hemophilia A and von Willebrand's disease, respectively. Each is a

glycoprotein, synthesized by different types of cells, and they associate extracellularly to form a complex. The FVIII-vWF complex is tightly, but noncovalently, associated. Because special measures must be taken to dissociate the complex, the two proteins tend to copurify. ThereThis work was supported by an American Heart Association fore, the now-established fact that FVIII and Established Investigator Award and by Grants HL 40921 vWF are distinct proteins was not widely acand HL 35058 from the National Institutes of Health, cepted until successful procedures were develPublic Health Service. oped to isolate functionally intact FVIII, free of Individual reprints of this article are not available. The vWF. entire Bowie Symposium on von Willebrand's Disease will Because vWF prolongs the circulatory lifebe available for purchase as a bound booklet from the time of FVIII and is probably necessary for the Proceedings Circulation Office in September. Mayo Clin Proc 66:524-534, 1991

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Mayo Clin Proc, May 1991, Vol 66

FACTOR VIII-voN WILLEBRAND FACTOR ASSOCIATION

normal expression of FVIII, severe von Willebrand's disease is associated with a secondary decrease in FVIII. The converse, however, is not true: severe hemophilia does not lead to a decrease in vWF. The factors that control the synthesis and expression of FVIII and vWF, their association, and the events that cause their dissociation are an active area of investigation. It is a tribute to Walter Bowie at the time of his retirement that, among his many areas of interest, the identification and characterization of porcine von Willebrand's disease by him and his colleagues have contributed substantially to our understanding of the biologic features of FVIII and vWF.

STRUCTURE AND FUNCTION OF FVIII FVIII has been isolated from bovine, porcine, and human plasma. By definition, these procedures involve a step that dissociates FVIII from vWF. This process can be accomplished by the addition of 0.25 M Mg2+, 0.25 M Ca2+, 1 M NaCI, or disulfide bond reducing agents. These procedures seem to have no effect on the functional properties of the FVIII molecule. Immediately after successful isolation of porcine 1and human" FVIII in sufficient amounts for amino-terminal sequencing, DNA probes were constructed for screening complementary DNA and genomic DNA libraries for the FVIII gene. The successful cloning of the entire FVIII gene" and isolation of a full-length complementary DNA encoding FVIII2,4,5 was a tour de force that resulted in background technology for the production of recombinant FVIII for use in the treatment of hemophilia A, valuable sequence information for structure-function studies, and developments in molecular biology important for the isolation of other large genes that are involved in human disease. The derived amino acid sequence from the full-length complementary DNA revealed that FVIII is synthesized as a single chain with three types of "domains" characterized by internal sequence homology in the following arrangement: AI-A2-B-A3-CI-C2 2 (Fig. 1). Subsequent cloning of a complementary DNA coding for

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factor V, which does not bind vWF, identified the same AI-A2-B-A3-CI-C2 sequence of domains," in which A- and C-type domains but not B domains are homologous in factor V and FVII!. Of particular interest is the presence in FVIII of two acidic regions, from residues 336 through 372 between the Al and A2 domains and from residues 1,648 through 1,689 between the Band A3 domains, that are not found in factor V. The former is necessary for procoagulant activity and is cleaved from FVIII and activated FVIII (FVIIIa) by activated protein C.8 The latter region is necessary for the association of FVIII with vWF (see subsequent discussion). Neither plasma-derived nor recombinant FVIII is isolated in single-chain form. Proteolysis at residue 1,648 results in the formation of a subunit called the FVIII light chain (FVIII Lc )' Additionally, proteolysis at undetermined sites within the B domain and between the A2 and B domains at residue 740 results in removal of some or all ofthe B domain (Fig. 1). Because the degree of heavy-chain proteolysis varies, several heavy-chain species arise. As a result, all reported FVIIIpreparations consist of a collection of heterodimers that contain a common light chain and a variably sized heavy chain, although the heterodimers can be separated to some extent by further chromatography.P!' Porcine FVIII is isolated from plasma as a mixture of three heterodimers with apparent molecular masses of 166/76, 130/76, and 82/76 kd. 1,5 Both plasma-derived and recombinant human FVIII have more heavy-chain heterogeneity than does porcine FVIIJ.2 The protease (or proteases) responsible for the cleavage of single-chain FVIII to its heterodimeric forms is not known, nor is it known whether FVIII circulates in single-chain form like factor V12 or undergoes proteolysis during the isolation procedure. Because intracellular proteolysis of human recombinant FVIII to typical heterodimeric forms occurs in Chinese hamster ovary cells during expression in vitro,13 most likely FVIII circulates predominantly in heterodimeric form. FVIII is activated by thrombin,14 factor Xa,8,15,16 and perhaps factor IXa and plasmin. The heterodimeric precursor forms of FVIII have little or

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FACTOR VIII-vON WILLEBRAND FACTOR ASSOCIATION

Mayo elin Proc, May 1991, Vol 66

Factor VIII NH 2

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Heavy Chain (HC)

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Synthesis + Processing 166 kDa ~ ~ 82 kDa ~ 1

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43 kDa

130 kDa

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The association of factor VIII with von Willebrand factor.

Factor VIII (FVIII) and von Willebrand factor (vWF) are plasma glycoproteins that circulate as a tightly associated complex. Because they tend to copu...
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